Neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease with dementia (PDD) and dementia with Lewy bodies (DLB) have no cure, but all three share signs of ?-synuclein (?-syn) pathology and of neuroinflammation. Although mounting evidence supports the notion that the immune cell response to neurodegeneration plays an active role in the pathogenesis of the above disorders, anti-inflammatory therapies have failed, thus far, to provide any beneficial effects in neurodegenerative diseases. We hypothesize that since immune cells exhibit a phenotypic heterogeneity, effective immune response-modifying therapy for neurodegenerative diseases requires the targeting of specific components of neuroinflammation rather than the broad inhibition of its signaling. The rationale for this project is that, once the regional and temporal genomic signatures of immune cells both inside and outside of the central nervous system (CNS) of animal models of synucleinopathies are known, meaningful biomarkers can be identified and innovative therapeutic strategies can be devised for PDD, DLB and even perhaps AD. Thus, the following two aims are proposed. To define the heterogeneity of the immune cell response to ?-syn pathology within the CNS, in AIM 1, we will define brain immune cell subpopulations in transgenic (Tg) mThy1-?-syn mouse line 61 (a recognized model of synucleinopathies) by droplet-based single-cell RNA-sequencing (scRNASeq) using freshly extracted immune cells from different brain regions at time points ranging from 2 (no/minimal neuroinflammation) to 14 months (overt neuroinflammation); non-Tg littermates will be used as controls. Using computational techniques adapted to low-depth single-cell RNA-seq data, we will identify transcriptomic subtypes of immune cells from the different mouse brain regions; this includes estimates of cluster robustness, characterization of cell type-specific genes, and identification of marker genes. In AIM1, we will also validate the single-cell data by probing for sets of newly- defined and existing marker genes in situ. Since ?-syn pathology also takes place outside of the CNS, in AIM 2, we will define the phenotypic diversity of the systemic immune cell response to ?-syn pathology in blood, spleen and lymph nodes from Tg Line 61 (and non-Tg littermates) at the same selected time points as in AIM 1, and will be subjected to the same scRNA-seq procedure and cell type analyses. We will then develop novel bi- compartmental models to relate changes in CNS and peripheral immune system cells, with the goal of identifying putative interactions between these two sets of cells in synucleinopathies. Successful completion of the proposed investigations will establish an atlas of immune cell phenotype heterogeneity in synucleinopathies and shed light into the cross talk between immune cells localized inside and outside of the CNS in response to ?-syn pathology. These findings will have an important positive impact on this area of research in that they will provide opportunities for biomarker identification and for therapeutic interventions and will advance our mechanistic understanding of PDD and DLB as well as possibly AD.